Prebiotic compositions comprising one or more types of bacteriophage

ABSTRACT

Disclosed herein are compositions that comprise one or more types of bacteriophage and methods of using such bacteriophages as a prebiotic to promote the growth of beneficial bacteria by decreasing harmful bacterial populations and releasing nutrients into the environment for good bacteria in the digestive system of an individual.

FIELD

Disclosed herein are compositions that comprise one or more types ofbacteriophage and methods of using such bacteriophages as a prebiotic topromote the growth of beneficial bacteria by decreasing harmfulbacterial populations and releasing nutrients into the environment forgood bacteria in the digestive system of an individual.

BACKGROUND

Microflora populations containing disproportionate concentrations ofundesirable organisms can be treated with antibiotics or cleansingregimes to eliminate most of the organisms in the gut, both desirableand undesirable, followed by probiotic supplement to reestablishmicroflora balance. High doses of probiotics, consumed in the form offermented foods containing the active probiotic organism or asnutritional supplements containing a specific minimum colony count ofthe probiotic are somewhat effective in controlling undesirableorganisms. To be effective, large doses of the probiotic must be takenregularly to establish and to maintain colonization and to overcome theadverse environment created by undesirable organisms (Handbook ofPrebiotics and Probiotics Ingredients, CRC Press, Boca Raton, Fla.2009).

To assist in establishing probiotic colonization in the presence oflarge established populations of undesirable organisms, prebiotics,which are usually non-digestible carbohydrates can be used. Thesecarbohydrate are selected for their ability to feed the probioticorganism preferentially and/or inhibit the growth or the undesirableorganism. Id.

Another mechanism which has been described for prebiotic function isinhibition of adhesion by undesirable microorganism to the intestinalwall (Quintero, M. I., “Adherence Inhibition of Cronobacter Sakazakiiand Other Pathogens By Prebiotic Oligosaccharides, Plant Extracts, andOther Naturally Derived Molecules,” Dissertations & Theses in FoodScience and Technology, University of Nebraska, Lincoln, Apr. 22, 2011).When adverse organisms are able to adhere and establish colonization,they modify the local environment to enhance its survival and to inhibitthe colonization by competing with desirable organisms. Certainnon-fermentable carbohydrates have structures resembling those found inthe surface of epithelial cells. These prebiotics bind to theundesirable bacteria preventing it from adhering to the intestinal walland allowing the organism to pass out of the digestive tract. Of courseonce the organism is attached to the intestinal wall, the prebiotic maybe ineffective in dislodging it.

The major products of probiotic metabolism are short chain fatty acids(SCFAs), the gases hydrogen and carbon dioxide, and bacterial cell mass.Unwanted symptoms relating to gas production in the gut are widelyreported in human prebiotic feeding studies including flatulence,bloating and diarrhea. Increased cell mass and unutilized prebioticcarbohydrate can produce an undesired laxative effect by stimulation ofperistalsis due to the increased bowel content (Gibson, G. R., et al.,“Selective Stimulation of Bifidobacteria in the Human Colon byOligofructose and Inulin,” Gastroenter 108:975-982, 1995).

In addition to polysaccharides, certain proteins and peptides and lipidsalso improve the growth of probiotics by inhibiting the undesirableorganisms. Examples are lactoferrin, proanthocyanidins, and a highmolecular weight component of cranberry juice. These materials may haveseveral mechanisms which inhibit colonization by undesirable organismsincluding inhibition of adherence. (Ofek I., et al., “Anti-adhesionTherapy of Bacterial Diseases,” FEMS Immunol Med Microbiol 38:181-191,2003.) These materials are quite expensive and require highconcentrations to be effective and their specificity may be more broadspectrum antibiotic than desired.

What are needed are new compositions for increasing the growth ofdesirable microflora. The methods and compositions disclosed hereinaccomplish this goal by administering one or more bacteriophage, whichlyse specific harmful and undesirable bacteria thus decreasing specificbacterial populations and thereby adding nutrients to the environmentfor the desirable bacteria.

SUMMARY

In accordance with the purposes of the disclosed materials, compounds,compositions, articles, and methods, as embodied and broadly describedherein, the disclosed subject matter, in one aspect, relates tocompositions and methods for preparing and using such compositions. In afurther aspect, the disclosed subject matter relates to compositionsused as a prebiotic to promote the growth of beneficial bacteria bydecreasing harmful bacteria populations and by releasing nutrientsextracted from the harmful bacteria into the environment, thusdecreasing bacterial crowding and providing nutrients from lysedunwanted bacteria for good bacteria in the digestive system of anindividual. The prebiotics used in the disclosed compositions andmethods are one or more bacteriophages, which have been discoveredherein to act as prebiotics.

Additional advantages will be set forth in part in the description thatfollows, and in part will be obvious from the description, or may belearned by practice of the aspects described below. The advantagesdescribed below will be realized and attained by means of the elementsand combinations particularly pointed out in the appended claims. It isto be understood that both the foregoing general description and thefollowing detailed description are exemplary and explanatory only andare not restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE FIGURES

The accompanying FIGURE, which is incorporated in and constitutes a partof this specification, illustrates several aspects described below.

FIG. 1 is a graph showing the optical density (a measure of celldensity) measured at intervals over 10 hours for various compositionsdisclosed herein.

DESCRIPTION

The materials, compositions, and methods described herein can beunderstood more readily by reference to the following detaileddescription of specific aspects of the disclosed subject matter and theExamples and FIGURE included herein.

Before the present materials, compositions, and methods are disclosedand described, it is to be understood that the aspects described beloware not limited to specific synthetic methods or specific reagents, assuch may, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularaspects only and is not intended to be limiting.

Also, throughout this specification, various publications arereferenced. The disclosures of these publications in their entiretiesare hereby incorporated by reference into this application in order tomore fully describe the state of the art to which the disclosed matterpertains. The references disclosed are also individually andspecifically incorporated by reference herein for the material containedin them that is discussed in the sentence in which the reference isrelied upon.

Definitions

In this specification and in the claims that follow, reference will bemade to a number of terms, which shall be defined to have the followingmeanings:

Throughout the specification and claims the word “comprise” and otherforms of the word, such as “comprising” and “comprises,” means includingbut not limited to, and is not intended to exclude, for example, otheradditives, components, integers, or steps.

As used in the description and the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a bacteriophage”includes mixtures of two or more such bacteriophages, reference to “anenzymes” includes mixtures of two or more such enzymes, reference to“the probiotic” includes mixtures of two or more such probiotics, andthe like.

“Optional” or “optionally” means that the subsequently described eventor circumstance can or cannot occur, and that the description includesinstances where the event or circumstance occurs and instances where itdoes not.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. “About” can mean within 5%of the stated value. When such a range is expressed, another aspectincludes from the one particular value and/or to the other particularvalue. Similarly, when values are expressed as approximations, by use ofthe antecedent “about,” it will be understood that the particular valueforms another aspect. It will be further understood that the endpointsof each of the ranges are significant both in relation to the otherendpoint, and independently of the other endpoint. It is also understoodthat there are a number of values disclosed herein, and that each valueis also herein disclosed as “about” that particular value in addition tothe value itself. For example, if the value “5” is disclosed, then“about 5” is also disclosed.

References in the specification and concluding claims to parts by weightof a particular element or component in a composition denotes the weightrelationship between the element or component and any other elements orcomponents in the composition for which a part by weight is expressed.Thus, in a compound containing 2 parts by weight of component X and 5parts by weight component Y, X and Y are present at a weight ratio of2:5, and are present in such ratio regardless of whether additionalcomponents are contained in the compound.

A weight percent (wt. %) of a component, unless specifically stated tothe contrary, is based on the total weight of the formulation orcomposition in which the component is included.

Reference will now be made in detail to specific aspects of thedisclosed materials, compounds, compositions, and methods, examples ofwhich are illustrated in the accompanying Examples and FIGURE.

Methods

Microbial biomass makes up over 50% of colonic contents. There are morethan 500 different culturable species of indigenous bacteria present inthe adult large intestine comprising around 1012 bacteria per gram dryweight (Moore et al., Some current concepts in intestinal bacteriology,Am. J. Clin. Nutr. 31:S33-42, 1978; Moore, et al., Intestinal flora inhealth and disease, Gastroenterology 86:174-193, 1984). Thesemicroorganisms perform a host of useful functions, such as fermentingunused energy substrates, training the immune system, preventing growthof harmful, pathogenic bacteria, regulating the development of the gut,producing vitamins for the host (such as biotin and vitamin K), andproducing hormones to direct the host to store fats. However, in certainconditions, some species are thought to be capable of causing disease byproducing infection or increasing cancer risk for the host. (Guarner etal., Gut flora in health and disease, Lancet 361(9356):512-519, 2003.)

It is widely recognized that maintaining a proper balance ofmicroorganisms in the intestine is essential to good health.Overpopulation of undesirable organisms modifies the intestinalenvironment to accommodate the undesirable species to the detriment ofdesirable organisms.

Probiotics are live microbial food supplements that beneficially affectthe host human or animal by improving its intestinal microbial balance.The benefits of probiotics, including displacement of undesirable orpathogenic organism, resistance to colonization of undesirable organismsby competitive exclusion and stimulation of the immune system, have beendocumented in numerous articles and patents. To be effective, probioticsare taken in large quantities and over extended periods of time.

Prebiotics are food ingredients that stimulate the growth and/oractivity of a probiotic in the digestive system so that the probioticcan provide the aforementioned benefits. Historically, prebiotics havebeen carbohydrates that are not digested directly by animals or humansbut that can be carbohydrate sources for specific probiotic organismsand not fermented as well by undesirable organisms. The concept is thatthese carbohydrates preferentially feed the probiotic organisms givingthem a competitive advantage over the undesirable organisms. To beeffective, large repetitive doses of the prebiotics are required. It isalso clear that large concentrations of other carbohydrates consumedwith meals would reduce the effectiveness of these prebiotics.

Disclosed herein are methods of using compositions that comprise one ormore lytic bacteriophages and one or more probiotic organisms. Thebacteriophage component of the disclosed compositions acts as prebioticto stimulate the growth of desirable organisms, including the probioticcomponent, as well as reduce the population of specific undesirablebacteria. The bacteriophage has specificity for one or more of theundesirable organisms. By weakening the population of the specificundesirable bacteria, the probiotic organisms can successfully competeand establish colonization producing an environment which is suitablefor them but inhospitable to undesirable bacteria. The specificundesirable bacteria are lysed and their cellular contents are availableas nutrients for probiotic organisms. No gasses or additional cellularmaterial are produced thus the aforementioned undesired side effects(flatulence, bloating, and diarrhea) are avoided. The bacteriophage arevery specific to the organism and as such do not directly affect anyother organisms in the digestive tract or any probiotic. The dose ofbacteriophage is quite small with no detectable impact on the materialbalance of the digestive process.

The disclosed methods can be achieved by administering a composition asdisclosed herein, and that comprises one or more lytic bacteriophagesand one or more probiotic organism. Further the disclosed methods cancomprise the administration of a nutrition composition as hereindescribed. Still further, disclosed herein is a method that comprisesadministering one or more lytic bacteriophages and then one or moreprobiotic organisms, or one or more probiotic organism and then one ormore lytic bacteriophages.

Compositions

Disclosed herein are compositions for increasing the growth of desirablebacteria and probiotic bacteria in the digestive system of anindividual. The disclosed compositions are nutritional compositions tobe administered orally to an individual and can also contain vitamins,minerals, and nutrients for the individual. The disclosed compositionscomprise one or more bacteriophages. These bacteriophages behave asprebiotics by stimulating the growth of the probiotic component of thedisclosed compositions. The bacteriophages reduce the population ofspecific undesirable bacteria including but not limited to bacteroides,coliforms, lysteria, helicobacter, salmonella and staphylococcus. Eachbacteriophage has specificity for an undesirable bacteria. By using thedisclosed bacteriophages, specific undesirable bacteria are lysed andtheir cellular material is available as nutrients for the probioticorganism or endogenous. Further, by weakening the population of thespecific undesirable bacteria, probiotic organisms can successfullycompete and establish colonization producing an environment which issuitable for them but inhospitable to the undesirable organism. As such,the disclosed compositions can comprise one or more probiotic organisms,as disclosed elsewhere herein. No gasses or extra cellular material areproduced; thus, the aforementioned undesired side effects are avoided.The bacteriophages of the disclosed compositions are very specific tothe undesirable bacteria and, as such, do not directly affect any otherorganisms in the digestive tract or any probiotic. The dose ofbacteriophage is quite small with no detectable impact on the materialbalance in the digestive process. Further by using the disclosedbacteriophages as a prebiotic, the use of more traditional prebioticssuch as polysaccharides can be avoided.

Bacteriophages Lytic bacteriophages are viruses that bind to specificbacterial cell surface receptors, inject their DNA, and take over thebiosynthetic machinery of the bacterium to produce daughter phages,which are released via host lysis to re-peat the process in other targetbacteria (Guttman et al., Basic phage biology. In: Bacteriophages:Biology and Applications. Kutter E and Sulakvelidze A (eds). New York:CRC Press, 2004, pp. 29-66). Bacteriophages have been isolated from manyenvironments, including the gastrointestinal tracts of food animals,where they are natural members of the microbial ecosystem (Orpin et al.,The occurrence of bacteriophages in the rumen and their influence onrumen bacterial population, Experientia 30:1018-1020, 1974; Klieve etal., Morphological diversity of ruminal bacteriophages from sheep andcattle, Appl Environ Microbiol 54(6):1637-1641, 1988). This disclosedcompositions comprise one or more lytic bacteriophages specificallytargeted to assist probiotic organism in overcoming populations ofharmful bacteria including but not limited to Bacteroides, Bordetella,Borrelia, Brucella, Campylobacter, Chlamydia and Chlamydophila,Clostridium, Corynebacterium, Enterococcus, Escherichia, Francisella,Haemophilus, Helicobacter, Legionella, Leptospira, Listeria,Mycobacterium, Mycoplasma, Neisseria, Pseudomonas, Rickettsia,Salmonella, Shigella, Staphylococcus, Streptococcus, Treponema, Vibrioand Yersinia.

In specific examples, the disclosed compositions comprise a lyticbacteriophage that is a member of the Bacteroidaceae family. In apreferred example, the bacteriophage is specific to Bacteroidesfragilis. The B. fragilis group includes B. fragilis (causes the mostclinical infections), Bacteroides distasonis, Bacteroides ovatus,Bacteroides thetaiotaomicron, and Bacteroides vulgatus. These bacteriaare resistant to penicillins, mostly through the production ofbeta-lactamase. They are part of the normal GI florae (Brook, I.,“Indigenous Microbial Flora of Humans,” In: Surgical InfectiousDiseases. 3rd Rd. Norwalk, Conn.: Appleton & Lange, 1995:37.)representing 30-50% of the fecal matter. They are responsible for 80% ofanaerobic intra-abdominal infections. Enterotoxigenic B. fragilis (ETBF)is also a potential cause of diarrhea. (Durmaz, B., et al., “Prevalenceof Enterotoxigenic Bacteroides fragilis in Patients with Diarrhea: aControlled Study,” Anaerobe 11(6):318-321, 2005.)

The Bacteroidaceae bacteriophages are part of the Siphoviridae family ofdouble-stranded DNA viruses infecting only bacteria. The virons arenonenveloped and have a long filamentous non-contractile tail and anisometric capsid (morphotype B1). The icosahedral capsid (T=7) is 57 nmin diameter. It is composed of 72 capsomers that appear hexagonal inoutline. The filamentous cross banded tail can reach to 570 nm. It has awidth of ˜8 nm and is non contractile. It has short terminal andsubterminal fibers. The genome is double stranded and linear. It istypically ˜50 kilobases in length and contains ˜70 genes. Theguanine+cytosine content is ˜52%.

In other examples, the disclosed compositions comprise a lyticbacteriophage that is specific to certain strains of Escherichia coli(E. coli). E. coli is a Gram-negative, rod-shaped bacterium that iscommonly found in the lower intestine of warm-blooded organisms makes upabout 0.1% of gut flora (Eckburg et al., Diversity of the HumanIntestinal Microbial Flora, Science. 14(308):1635-1638, 2005). Most E.coli strains are harmless, but some serotypes can cause serious foodpoisoning in humans. Virulent strains of E. coli can causegastroenteritis, urinary tract infections, and neonatal meningitis. Inrare cases, virulent strains are also responsible for hemolytic-uremicsyndrome, peritonitis, mastitis, septicemia and Gram-negative pneumonia.

Rakieten et al., Studies with bacteriophages active against mucoidstrains of bacteria, J. Bacteriol. 40:529-545, 1940; Demerec et al.,Bacteriophage-resistant mutants in Escherichia coli, Genetics30:119-136, 1944. Delbrück, Bacterial viruses or bacteriophages, Biol.Rev. 21:30-40, 1946.

In the disclosed compositions and methods, a bacteriophage is used,which is not the same component as a lytic protein isolated from abacteriophage.

Probiotic Organisms

The probiotic component of the disclosed compositions comprises livemicroorganisms that beneficially affect the host individual bydisplacement of undesirable or pathogenic bacteria and by creating anenvironment that resists colonization of the undesirable bacteria. Whencolonization of undesirable bacteria is present, probiotic organismsmust compete with the established colonies. The combination of abeneficial bacteria with the disclosed bacteriophages can produce asymbiotic mixture.

In specific examples, the disclosed compositions comprise a probioticorganism that is a Lactobacillus species, such as L. acidophilus, L.amylovorus, L. brevis, L. casei, L. casei subsp. rhamnosus(Lactobacillus GG), L. caucasicus, L. crispatus, L. delbrueckii subsp.bulgaricus (L. bulgaricus), L. fermentum (L. fermenti), L. gasseri, L.helveticus, L. johnsonii, L. lactis, L. leichmannii, L. paracasei, L.plantarum, L. reuteri, and L. rhamnosus. In other examples, thedisclosed compositions comprise a probiotic organism that is aBiifidobacterium species, such as B. adolescentis, B. bifidum, B. breve,B. infantis, B. lactis (B. animalis), B. licheniformis, and B. longum.In still other examples, the disclosed compositions comprise a probioticorganism that is a lactic acid bacteria such as Enterococcus faecium,Lactococcus lactis, Leuconstoc mesenteroides, Pediococcus acidilactici,Streptococcus thermophilus). In yet other examples, the disclosedcompositions comprise a probiotic organism that is a nonlactic acidbacteria such as Bacillus subtilis, Saccharomyces boulardii, andSaccharomyces cerevisiae.

Additional Components

In further embodiments, the disclosed compositions can comprise othertypes of prebiotics. For example, the disclosed compositions cancomprise inulin, fructooligosaccharides, galactooligiosaccharides,xylooligosaccharides, polydextrose, lactulose, tagatose,isomaltooligosaccarides, soybean oligosaccharides, lactoferrin, andproanthocyanins. Though with the use of the disclosed bacteriophages,which act themselves as prebiotics, other prebiotics are not necessary.

The disclosed compositions can also include nutritional supplements,such as vitamins, minerals, trace elements, polyunsaturated fatty acids,antioxidants, amino acids, and the like.

Specific Combinations

Specific examples of suitable compositions disclosed herein include abacteriophage that is specific to Bacteroides fragilis and/or E. coliand a probiotic selected from the group consisting of Lactobacillus (L.)acidophilus, L. amylovorus, L. brevis, L. casei, L. casei subsp.rhamnosus (Lactobacillus GG), L. caucasicus, L. crispatus, L.delbrueckii subsp. bulgaricus (L. bulgaricus), L. fermentum (L.fermenti), L. gasseri, L. helveticus, L. johnsonii, L. lactis, L.leichmannii, L. paracasei, L. plantarum, L. reuteri, and L. rhamnosus.In another specific example, the disclosed compositions can comprise abacteriophage that is specific to Bacteroides fragilis and/or E. coliand a probiotic selected from the group consisting of Bifidobacterium(B.) adolescentis, B. bifidum, B. breve, B. infantis, B. lactis (B.animalis), B. licheniformis, and B. longum. In another specific example,the disclosed compositions can comprise a bacteriophage that is specificto Bacteroides fragilis and/or E. coli and a probiotic selected from thegroup consisting of Enterococcus faecium, Lactococcus lactis, Leuconstocmesenteroides, Pediococcus acidilactici, and Streptococcus thermophilus.In another specific example, the disclosed compositions can comprise abacteriophage that is specific to Bacteroides fragilis and/or E. coliand a probiotic selected from the group consisting of Bacillus subtilis,Escherichia coli strain nissle, Saccharomyces boulardii, andSaccharomyces cerevisiae.

Nutritional Compositions, Administration, Dosage

As described, the compositions disclosed herein can be provided in anutritional composition. Depending on the intended mode ofadministration, the nutritional composition can be in the form of solid,semi-solid or liquid dosage forms, such as, for example, tablets, pills,capsules, powders, liquids, or suspensions, preferably in unit dosageform suitable for single administration of a precise dosage. Thecompositions will include an effective amount of the compositionsdescribed herein optionally in combination with a biologicallyacceptable carrier and, in addition, can include other flavorings,thickeners, chelating agents, binders, carriers, or diluents. By“biologically acceptable” is meant a material that is not biologicallyor otherwise undesirable, which can be administered to an individualalong with the selected compositions without causing unacceptablebiological effects or interacting in a deleterious manner with the othercomponents of the nutritional composition in which it is contained.

As used herein, the term carrier encompasses any excipient, diluent,filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, orother material well known in the art for use in nutritionalformulations. The choice of a carrier for use in a composition willdepend upon the intended route of administration for the composition.The preparation of nutritional acceptable carriers and formulationscontaining these materials is described in, e.g., Remington'sPharmaceutical Sciences, 21st Edition, ed. University of the Sciences inPhiladelphia, Lippincott, Williams & Wilkins, Philadelphia Pa., 2005.Examples of biologically acceptable carriers include buffers such asphosphate buffers, citrate buffer, and buffers with other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptides; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™ (ICI, Inc.; Bridgewater, N.J.), polyethylene glycol(PEG), and PLURONICS™ (BASF; Florham Park, N.J.). Further examples ofsuitable aqueous and nonaqueous carriers, diluents, solvents or vehiclesinclude water, ethanol, polyols (propyleneglycol, polyethyleneglycol,glycerol, and the like), suitable mixtures thereof, vegetable oils (suchas olive oil) and animal oils (fish oil). Proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionsand by the use of surfactants.

These compositions can also contain adjuvants such as preserving,wetting, emulsifying, and dispensing agents. Isotonic agents, forexample, sugars, sodium chloride, and the like can also be included.

Solid dosage forms for oral administration of the compositions describedherein include capsules, tablets, pills, powders, and granules. In suchsolid dosage forms, the compositions described herein is admixed with atleast one inert customary excipient (or carrier) such as sodium citrateor dicalcium phosphate or (a) fillers or extenders, as for example,starches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders, as for example, carboxymethylcellulose, alignates, gelatin,polyvinylpyrrolidone, sucrose, and acacia, (c) humectants, as forexample, glycerol, (d) disintegrating agents, as for example, agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certaincomplex silicates, and sodium carbonate, (e) solution retarders, as forexample, paraffin, (f) absorption accelerators, as for example,quaternary ammonium compounds, (g) wetting agents, as for example, cetylalcohol, and glycerol monostearate, (h) adsorbents, as for example,kaolin and bentonite, and (i) lubricants, as for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules, tablets, andpills, the dosage forms can also comprise buffering agents.

Solid compositions of a similar type can also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols, andthe like.

Solid dosage forms such as tablets, dragées, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others known in the art. They can contain opacifying agentsand can also be of such composition that they release the activecompound or compounds in a certain part of the intestinal tract in adelayed manner. Examples of embedding compositions that can be used arepolymeric substances and waxes. The active compounds can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-mentioned excipients.

Liquid dosage forms for oral administration of the compositionsdescribed herein include pharmaceutically acceptable emulsions,solutions, suspensions, syrups, and elixirs. In addition to the activecompounds, the liquid dosage forms can contain inert diluents commonlyused in the art, such as water or other solvents, solubilizing agents,and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate,propyleneglycol, 1,3-butyleneglycol, dimethylformamide, oils, inparticular, cottonseed oil, groundnut oil, corn germ oil, olive oil,castor oil, sesame oil, glycerol, tetrahydrofurfuryl alcohol,polyethyleneglycols, and fatty acid esters of sorbitan, or mixtures ofthese substances, and the like.

Besides such inert diluents, the composition can also include additionalagents, such as wetting, emulsifying, suspending, sweetening, flavoring,or perfuming agents.

Suspensions, in addition to the active compounds, can contain additionalagents, as for example, ethoxylated isostearyl alcohols, polyoxyethylenesorbitol and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, or mixtures of thesesubstances, and the like.

Administration of the compositions described herein to an individual canbe carried out using effective amounts of the compositions describedherein for periods of time effective to treat a disease or infection. Anindividual can include both mammals and non-mammals. Mammals include,for example, humans; nonhuman primates, e.g. apes and monkeys; cattle;horses; sheep; rats; mice; pigs; and goats. Non-mammals include, forexample, fish and birds. In a preferred embodiment, the individual is ahuman.

The effective amount of the compositions described herein can bedetermined by one of ordinary skill in the art and includes exemplarydosage amounts for a mammal of from about 0.5 to about 200 mg/kg of bodyweight of active compound per day, which can be administered in a singledose or in the form of individual divided doses, such as from 1 to 4times per day. Alternatively, the dosage amount can be from about 0.5 toabout 150 mg/kg of body weight of active compound per day, about 0.5 to100 mg/kg of body weight of active compound per day, about 0.5 to about75 mg/kg of body weight of active compound per day, about 0.5 to about50 mg/kg of body weight of active compound per day, about 0.5 to about25 mg/kg of body weight of active compound per day, about 1 to about 20mg/kg of body weight of active compound per day, about 1 to about 10mg/kg of body weight of active compound per day, about 20 mg/kg of bodyweight of active compound per day, about 10 mg/kg of body weight ofactive compound per day, or about 5 mg/kg of body weight of activecompound per day. The expression effective amount, when used to describean amount of compound in a method, refers to the amount of a compoundthat achieves the desired pharmacological effect or other effect, forexample an amount that results in bacterial inhibition or growth.

For the bacteriophage component of the disclosed compositions, they canbe administered in an effective amount of from 1 colony forming unit(CFU)/mL to 1×10¹³ plaque forming unit (PFU)/mL. 1 PFU/mL isapproximately 1 phage/mL or 1 phage/g. In certain examples thebacteriophage component of the disclosed compositions can beadministered in amounts of from about 1×10² to about 1×10¹³, from about1×10⁴ to about 1×10¹¹, from about 1×10⁶ to about 1×10¹⁰, from about1×10³ to about 1×10⁹, from about 1×10⁵ to about 1×10⁷, or from about1×10⁷ to about 1×10¹¹ PFU/mL or PFU/g. In further examples thebacteriophage can be administered in amounts of from about 1×10⁴ toabout 1×10⁶ PFU/mL or PFU/g. In still further examples, thebacteriophage component can be administered in amounts of at least about1×10¹, 1×10², 1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰,1×10¹¹, 1×10¹², or 1×10¹³ PFU/mL or PFU/g.

Those of skill in the art will understand that the specific dose leveland frequency of dosage for any particular subject can be varied andwill depend upon a variety of factors, including the activity of thespecific composition employed, the metabolic stability and length ofaction of that composition, the species, age, body weight, generalhealth, sex and diet of the subject, the mode and time ofadministration, rate of excretion, active combination, and severity ofthe particular condition.

The examples below are intended to further illustrate certain aspects ofthe methods and compounds described herein, and are not intended tolimit the scope of the claims.

EXAMPLES

The following examples are set forth below to illustrate the methods andresults according to the disclosed subject matter. These examples arenot intended to be inclusive of all aspects of the subject matterdisclosed herein, but rather to illustrate representative methods andresults. These examples are not intended to exclude equivalents andvariations that are apparent to one skilled in the art.

Efforts have been made to ensure accuracy with respect to numbers (e.g.,amounts, temperature, etc.) but some errors and deviations should beaccounted for. Unless indicated otherwise, parts are parts by weight,temperature is in ° C. or is at ambient temperature, and pressure is ator near atmospheric. There are numerous variations and combinations ofreaction conditions, e.g., component concentrations, temperatures,pressures and other reaction ranges and conditions that can be used tooptimize the product purity and yield obtained from the disclosedprocess. Only reasonable and routine experimentation will be required tooptimize such process conditions.

In vitro studies to determine the ability of bacteriophage to allowspecific changes, both in the composition and/or activity ingastrointestinal microflora, were done under physiological conditions ofthe small intestine (37° C. and pH 6.8). The bacteria selected fortesting included three species of Bacteroides, namely strains offragilis, uniformis and thetaiotaomicron and two species of E. coli,namely strains of E. coli B and C and five strains of probiotics:Bacillus subtilis, Lactobacillus paracasei, Lactobacillus lactis,Bifidobacterium breve and Bifidobacterium longum.

Example 1

Bacteroides fragilis, Bacteroides uniformis, Bacteroidesthetaiotaomicron and four strains of probiotics; Lactobacillusparacasei, Lactobacillus lactis, Bifidobacterium breve andBifidobacterium longum (all Bacteroides strains purchased from ATCC)were tested in vitro. Frozen permanents of each bacterial strain werestreaked for single colonies using Bile Esculin agar or nutrient agarwarmed prior to use. For fresh inoculants, single bacterial cells werere-streaked on a Bile Esculin agar. The bacteriophage was propagatedfrom bacteroides fragilis after 16-20 hours of bacterial growth, 16000rpm cell clearing spin for 30 minutes and collection of the supernatant.Bacteriophage were concentrated using an Amicon Ultra-15.50 kDa cutoffconcentrator, titers were determined by counting plaques from serialdilutions using the bacterial host strain. The Bacteroides fragilisbacteriophage (FBP) Cocktail was diluted to 1×10⁶ PFU/mL.

Bacterial cells of several fresh colonies were incubated in 50 mLs ofnutrient broth (1% glucose) until the culture reached an optical densityof 0.2. For anaerobic growth, 0.2 cultures were divided into flasks andpurged with a 3-5 second shot of nitrogen, bacteriophage were quicklyadded at a multiplicity of infection (MOI) of 1 to one set and broth tothe other for a control to each flask, sealed and put back on theshaker. Anaerobic samples were opened once, tested and discarded. Theoptical density was measured at intervals over 10 hours. See FIG. 1.

The addition of the FBP to the Bacteroides fragilis culture rapidlyreduced the population while the addition of FBP had no effect on any ofthe other organisms tested.

Example 2

Bacteroides fragilis and four strains of probiotics; Lactobacillusparacasei, Lactobacillus lactis, Bifidobacterium breve andBifidobacterium longum (all Bacteroides strains purchased from ATCC)were tested in vitro. Bacteroides is the dominate bacteria in the humangut, while most of the time, most of the strains of Bacteroides aresymbiotic, certain bacteroides strains can be pathogenic. One of themain bacteroides strains known to cause pathogenicity in humans isbacteroides fragilis, which can take over the gastrointestinal tractrobbing nutrients and crowding out good bacteria. For that reason, aprebiotic bacteriophage was chosen for its ability to quickly andefficiently lyse Bacteroides fragilis exclusively, providing goodbacteria nutrients while maintaining a healthy natural gut flora. Frozenpermanents of each bacterial strain were streaked for single coloniesusing Bile Esculin agar or nutrient agar warmed prior to use. For freshinoculants, single bacterial cells were re-streaked on a Bile Esculinagar. The bacteriophage was propagated from Bacteroides fragilis after16-20 hours of bacterial growth, 16000 rpm cell clearing spin for 30minutes and collection of the supernatant. Bacteriophage wereconcentrated using an Amicon Ultra-15.50 kDa cutoff concentrator, titerswere determined by counting plaques from serial dilutions using thebacterial host strain. The Bacteroides fragilis bacteriophage (FBP)Cocktail was diluted to 1×10⁶ PFU/mL.

Bacterial cells of several fresh colonies were incubated in 50 mLs ofnutrient broth (1% glucose) until the culture reached an optical densityof 0.2. For anaerobic growth, 0.2 cultures were divided into flasks andpurged with a 3-5 second shot of nitrogen, bacteriophage were quicklyadded at a multiplicity of infection (MOI) of 1 to one set and broth tothe other for a control to each flask, sealed and put back on theshaker. Anaerobic samples were opened once, tested and discarded.Probiotic alone, with bacteroides, with bacteroides and FBP cocktailtogether and with FBP cocktail were mixed and allowed to grow forindicated time period in a shaker bath at 37° C. and pH 6.8. Onemilliliter samples were taken and diluted several times in HardyDiagnostic phosphate buffer and plated on selected media for each typeof probiotic and for bacteroides on bacteroides bile esculin plates (PMLmicrobiologicals) and the selected media alone as a control. Bacteroidesthat grew on the selected media for the probiotic was subtracted outfrom the total plate count of the mixture. MRS agar (Neogen) plates wereused for quantification of Lactobacillus strains, Bifidobacterium agar(Hardy Diagnostics) were used for the quantification of Bifidobacteriumstrains. Experiments were done in triplicate, multiple dilutions weredone and 5 or more plates were averaged to determine bacterial counts.Test counts were subtracted out from control plates for final samplecounts. See Table 1.

TABLE 1 Nutrient Broth CFU/mL after 5 hr Bacteroides Strains L.Paracasei Fragilis Individual (anaerobic) 1 × 10³ 4 × 10³ L. Paracasei +Bacteroides Fragilis 2 × 10² 1 × 10² L. Paracasei + BacteroidesFragilis + 7 × 10³ 5 FBP Bacteroides Strains L. Lactis FragilisIndividual (anaerobic) 9 × 10³ 1 × 10⁴ L. Lactis + Bacteroides Fragilis7 × 10² 3 × 10³ L. Lactis + Bacteroides Fragilis + 2 × 10⁴ 0 FBPBacteroides Strains B. Longum Fragilis Individual (anaerobic) 2 × 10³ 6× 10³ B. Longum + Bacteroides Fragilis 1 × 10² 1 × 10³ B. Longum +Bacteroides Fragilis + 7 × 10³ 12 FBP Bacteroides Strains B. BreveFragilis Individual (anaerobic) 1 × 10⁴ 9 × 10³ B. Breve + BacteroidesFragilis 4 × 10² 7 × 10³ B. Breve + Bacteroides Fragilis + 5 × 10⁴ 6 FBP

Mixed cultures of Bacteroides fragilis and each of the probioticsreduced the growth of both the probiotic and of the Bacteroides ascompared with the pure cultures. The addition of FPB cocktailessentially eliminated the Bacteroides fragilis while significantlyincreasing the growth of each probiotic over the controls. These resultsindicate that the lytic activity on the Bacteroides provides nutrientswhich stimulate the growth of each of the probiotics tested.

Example 3

Bacterial cells of several fresh colonies were incubated in 50 mLs ofnutrient broth (1% glucose) until the culture reached an optical densityof 0.2. The bacteria was spun down and re-suspended in minimal mediawith 1% glucose, 1% inulin or 1% Isomalta-oligosaccharide as the solecarbon source. For anaerobic growth, 0.2 cultures were divided intoflasks and purged with a 3-5 second shot of nitrogen, bacteriophage werequickly added at a multiplicity of infection (MOI) of 1 to one set andbroth to the other for a control to each flask, sealed and put back onthe shaker. Anaerobic samples were opened once, tested and discarded.Probiotic alone, with bacteroides, with bacteroides and FBP cocktailtogether and with FBP cocktail were mixed and allowed to grow forindicated time period in a shaker bath at 37° C. and pH 6.8. Onemilliliter samples were taken and diluted several times in HardyDiagnostic phosphate buffer and plated on selected media for each typeof probiotic and for bacteroides on bacteroides bile esculin plates (PMLmicrobiologicals) and the selected media alone as a control. Bacteroidesthat grew on the selected media for the probiotic was subtracted outfrom the total plate count of the mixture. MRS agar (Neogen) plates wereused for quantification of Lactobacillus, Bifidobacterium agar (HardyDiagnostics) were used for the quantification of Bifidobacterium.Experiments were done in triplicate, multiple dilutions were done and 5or more plates were averaged to determine bacterial counts. Test countswere subtracted out from control plates for final sample counts. SeeTable 2.

TABLE 2 MM + Inulin CFU/mL after 48 hr Bacteroides Strain(s) L.Paracasei Fragilis Individual (anaerobic) 2730 0 L. Paracasei +Bacteroides Fragilis 2850 0 L. Paracasei + Bacteroides Fragilis + 2650 0FBP Bacteroides Strain(s) B. Longum Fragilis Individual (anaerobic) 25900 B. Longum + Bacteroides Fragilis 2340 0 B. Longum + BacteroidesFragilis + 2570 0 FBP MM + Vitafiber CFU/mL after 48 hr BacteroidesStrain(s) L. Paracasei Fragilis Individual (anaerobic) 3610 1240 L.Paracasei + Bacteroides Fragilis 2780 970 L. Paracasei + BacteroidesFragilis + 3830 0 FBP Bacteroides Strain(s) B. Longum FragilisIndividual (anaerobic) 3980 1060 B. Longum + Bacteroides Fragilis 2790780 B. Longum + Bacteroides Fragilis + 4120 0 FBP Nutrient + InulinCFU/mL after 48 hr Bacteroides Strain(s) L. Paracasei FragilisIndividual (anaerobic) 1 × 10⁶ 9 × 10⁴ L. Paracasei + BacteroidesFragilis 9 × 10³ 6 × 10² L. Paracasei + Bacteroides Fragilis + 8 × 10⁶16 FBP Bacteroides Strain(s) B. Longum Fragilis Individual (anaerobic) 7× 10⁶ 8 × 10⁴ B. Longum + Bacteroides Fragilis 2 × 10⁴ 1 × 10³ B.Longum + Bacteroides Fragilis + 1 × 10⁷ 23 FBP Nutrient + VitafiberCFU/mL after 48 hr Bacteroides Strain(s) L. Paracasei FragilisIndividual (anaerobic) 3 × 10⁶ 6 × 10⁵ L. Paracasei + BacteroidesFragilis 4 × 10⁴ 5 × 10³ L. Paracasei + Bacteroides Fragilis + 4 × 10⁷10 FBP Bacteroides Strain(s) B. Longum Fragilis Individual (anaerobic) 1× 10⁶ 3 × 10⁵ B. Longum + Bacteroides Fragilis 1 × 10⁴ 7 × 10³ B.Longum + Bacteroides Fragilis + 8 × 10⁶ 8 FBP

The results indicate that the bacteriophage had no negative effect oneither the Lactobacillus or Bifidobacterium. Neither inulin norIsomalta-oligosaccharide alone were good nutrients for any of theorganisms. In nutrient broth with either inulin orIsomalta-oligosaccharide added the Bacteroides and probiotics inhibiteach other's growth. In nutrient broth with either inulin orIsomalta-oligosaccharide added, in the presence of the bacteriophage,the Bacteroides fragilis population is reduced by several logs while theprobiotic population is increased about 3 logs and about one log overthe probiotic controls. This corresponds to the previous experiment andprovides further evidence that the lysed bacteroides is being used bythe probiotics as nutrient.

Example 4

Four bacteriophage were chosen for their ability to quickly andefficiently lyses several strains of E. coli including E. coli B, fiveEnterotoxigenic Escherichia coli, or ETEC, that produces heat stabletoxin (ST), five ETEC E. coli strains that produce heat labile (LT),five that produce both ST and LT toxins and E. coli O157:H7.

The bacteriophage were propagated from non-pathogenic E. coli B, C orK12 (all strains were a gift from Texas A&M University) after 12-16hours of bacterial growth, 16000 rpm cell clearing spin for 30 minutesand collection of the supernatant. Bacteriophage were concentrated usingan Amicon Ultra-15.50 kDa cutoff concentrator, titers were determined bycounting plaques from serial dilutions using the bacterial host strain.The E. coli bacteriophage Cocktail (EBP) were diluted to 1×10⁶ PFU/mLand mixed at a 1:1:1:1 ratio.

Bacterial cells of several fresh colonies were incubated in 50 mLs ofnutrient broth (1% glucose) until the culture reached an optical densityof 0.2. For competition experiments, each bacteria was grown to an O.D.of 0.2, halved and added together. Bacteriophages were quickly added ata multiplicity of infection (MOI) of 1 to one set and broth to the otherfor a control. For anaerobic growth, 0.2 cultures were divided into 9flasks and purged with a 5 second shot of nitrogen, bacteriophage(MOI 1) and broth were divided up and added to each flask, sealed andput back on the shaker. Anaerobic samples were opened once, tested anddiscarded. Probiotic alone, probiotic with E. coli, probiotic with bothE. coli and EBP together and probiotic with only EBP were mixed andallowed to grow for 1 hour in a shaker bath at 37° C. and pH 6.8. Onemilliliter samples were taken and diluted several times in HardyDiagnostic phosphate buffer and plated on selected media for each typeof probiotic and for E. coli on 3M E. coli plates and the selected mediaalone as a control. E. coli that grew on the selected media for theprobiotic was subtracted out from the total plate count of the mixture.Nutrient agar (Hardy Diagnostics) plates were used for quantification ofBacillus subtilis, MRS agar (Neogen) plates were used for quantificationof Lactobacillus paracasei and Lactobacillus lactis, Bifidobacteriumagar (Hardy Diagnostics) were used for the quantification ofBifidobacterium breve and Bifidobacterium longum. Experiments were donein triplicate, multiple dilutions were done and 5 or more plates wereaveraged to determine bacterial counts.

Example 4a

Bacillus subtilis was evaluated as described above both aerobically andanaerobically. Under aerobic conditions, E. coli reduced the probioticby almost 2 logs. With EPB added the E. coli was reduced by 10 logswhile allowing the probiotic to increase 2 logs. Under anaerobic E. colireduced the probiotic by 1 log. With EPB added E. coli was reduced by 1log while allowing the probiotic to increase almost 2 logs (Table 3).

TABLE 3 Log CFU Bacillus Subtilis E. coli B Individual (aerobic) 7.3 9.8B. Subtilis + E. coli B(aerobic) 5.6 7.8 B. Subtilis + E. coli B +EBP(aerobic) 7.7 0.0 Individual (anaerobic) 3.0 4.5 B. Subtilis + E.coli B(anaerobic) 1.9 3.0 B. Subtilis + E. coli B + EBP(anaerobic) 3.62.0

Example 4b

Lactobacillus paracasei was evaluated as described above bothaerobically and anaerobically. Under aerobic conditions, E. coli reducedthe probiotic by almost 3 logs. With EPB added the E. coli was reducedby 7 logs while allowing the probiotic to increase 1 log. Underanaerobic E. coli reduced the probiotic by over 1 log. With EPB added E.coli was reduced by over 2 log while allowing the probiotic to increasealmost 1 log (Table 4).

TABLE 4 Log CFU L. Paracasei E. coli B Individual (aerobic) 5.5 9.0 L.Paracasei + E. coli B (aerobic) 2.8 7.0 L. Paracasei + E. coli B + EBP(aerobic) 4.0 0.5 Individual (anaerobic) 3.0 6.0 L. Paracasei + E. coliB (anaerobic) 1.5 5.0 L. Paracasei + E. coli B + EBP (anaerobic) 2.8 2.5

Example 4c

Lactobacillus lactis was evaluated as described above anaerobically. E.coli reduced the probiotic by almost 2 logs. In presence of the EPB theE. coli was reduced by 3 logs while allowing the probiotic to increase 1log (Table 5).

TABLE 5 Log CFU L. Lactis E. coli B Individual (anaerobic) 3.8 5.9 L.Lactis + E. coli B 2.0 4.0 L. Lactis + E. coli B + EBP 3.0 1.0

Example 4d

Bifidobacterium longum was evaluated as described above anaerobically.E. coli reduced the probiotic by almost 2 logs. In presence of the EPBthe E. coli was reduced by 3 log while allowing the probiotic toincrease 1 log (Table 6).

TABLE 6 Log CFU B. Longum E. coli B Individual (anaerobic) 4.3 5.9 B.Longum + E. coli B 2.5 4.3 B. Longum + E. coli B + EBP 3.8 1.0

Example 4e

Bifidobacterium Breve was evaluated as described above anaerobically. E.coli reduced the probiotic by almost 2 logs. In presence of the EPB theE. coli was reduced by 2 log while allowing the probiotic to increase 1log (Table 7).

TABLE 7 Log CFU B. Breve E. coli B Individual (anaerobic) 4.5 5.9 B.Breve + E. coli B 2.8 4.7 B. Breve + E. coli B + EBP 4.0 2.3

In each example, the E. coli population inhibited the growth of theprobiotic organism. Also in every case, the addition of EPB reduced theE. coli by more than 98% while increasing the probiotic population bymore than 10 fold.

Clearly the use of EPB either as a symbiotic mixture or used separatelybut simultaneously provides a significant improvement in probioticgrowth.

The compositions and methods of the appended claims are not limited inscope by the specific compositions and methods described herein, whichare intended as illustrations of a few aspects of the claims and anycompositions and methods that are functionally equivalent are within thescope of this disclosure. Various modifications of the compositions andmethods in addition to those shown and described herein are intended tofall within the scope of the appended claims. Further, while onlycertain representative compositions, methods, and aspects of thesecompositions and methods are specifically described, other compositionsand methods and combinations of various features of the compositions andmethods are intended to fall within the scope of the appended claims,even if not specifically recited. Thus a combination of steps, elements,components, or constituents can be explicitly mentioned herein; however,all other combinations of steps, elements, components, and constituentsare included, even though not explicitly stated.

1-13. (canceled)
 14. A method for increasing the growth of probioticorganisms in the digestive system of an individual, comprisingadministering a composition comprising one or more lytic bacteriophagesspecific to Escherichia family and one or more probiotic organismschosen from Lactobacillus species and Bifidobacterium species.
 15. Themethod of claim 14, wherein the composition comprises one or moreadditional bacteriophages specific to Bacteroides, Bordetella, Borrelia,Brucella, Campylobacter, Chlamydia and Chlamydophila, Clostridium,Corynebacterium, Enterococcus, Escherichia, Francisella, Haemophilus,Helicobacter, Legionella, Leptospira, Listeria, Mycobacterium,Mycoplasma, Neisseria, Pseudomonas, Rickettsia, Salmonella, Shigella,Staphylococcus, Streptococcus, Treponema, Vibrio and Yersinia.
 16. Themethod of claim 15, wherein the additional bacteriophage is specific toBacteroides distasonis, Bacteroides ovatus, Bacteroidesthetaiotaomicron, and Bacteroides vulgates, or Bacteroides fragilis. 17.The method of claim 14, wherein the bacteriophage is specific to E. coliB, Enterotoxigenic E. coli (ETEC), or E. coli O157:H7.
 18. The method ofclaim 14, wherein the probiotic organism is a Lactobacillus species. 19.The method of claim 14, wherein the probiotic organism is L.acidophilus, L. amylovorus, L. brevis, L. casei, L. casei subsp.rhamnosus (Lactobacillus GG), L. caucasicus, L. crispatus, L.delbrueckii subsp. bulgaricus (L. bulgaricus), L. fermentum (L.fermenti), L. gasseri, L. helveticus, L. johnsonii, L. lactis, L.leichmannii, L. paracasei, L. plantarum, L. reuteri, or L. rhamnosus.20. The method of claim 14, wherein the probiotic organism is aBiifidobacterium species.
 21. The method of claim 14, wherein theprobiotic organism is one or more B. adolescentis, B. bifidum, B. breve,B. infantis, B. lactis (B. animalis), B. licheniformis, and B. longum.22. The method of claim 14, wherein the probiotic organism is one ormore Enterococcus faecium, Lactococcus lactis, Leuconstoc mesenteroides,Pediococcus acidilactici, and Streptococcus thermophilus.
 23. The methodof claim 14, wherein the probiotic organism is Bacillus subtilis,Saccharomyces boulardii, or Saccharomyces cerevisiae.
 24. The method ofclaim 1, wherein the composition further comprises comprise inulin,fructooligosaccharides, galactooligiosaccharides, xylooligosaccharides,polydextrose, lactulose, tagatose, isomaltooligosaccarides, soybeanoligosaccharides, lactoferrin, and proanthocyanins.
 25. The method ofclaim 14, wherein the amount of bacteriophage in the composition is atleast 1×10⁴ CFU/g. 26-38. (canceled)